安宫牛黄丸治疗中重度急性缺血性卒中的安全性和有效性（ANGONG TRIAL）：一项随机双盲安慰剂对照的先导临床试验
抽象的
背景：
临床前研究表明，安宫牛黄丸（ANP）可减少脑梗死和脑水肿体积。本研究旨在探讨ANP能否安全地减少中重度急性缺血性卒中患者的脑梗死和脑水肿体积。
方法：
这项随机、双盲、安慰剂对照的先导试验纳入了2021年4月至2022年7月期间在中国17个中心确诊的急性缺血性卒中患者，这些患者的美国国立卫生研究院卒中量表(NIHSS)评分为10至20分。患者在发病后36小时内通过区组随机分组，分别接受ANP或安慰剂治疗（每日3克，共5天）。主要结局指标是治疗14天后脑梗死和水肿体积的变化。主要安全性结局指标是90天内严重不良事件(SAE)。
结果：
最终分别有57例和60例患者被纳入ANP组进行改良意向治疗分析。中位年龄为66.0岁，基线NIHSS评分中位数为12.0分。ANP组和安慰剂组第14天脑梗死体积变化量分别为0.3 mL和0.4 mL（中位数差：-7.1 mL，四分位距[IQR]：-18.3～2.3 mL，P =0.30）。ANP组和安慰剂组第14天脑水肿体积变化量分别为11.4 mL和4.0 mL（中位数差：3.0 mL，IQR：-1.3～9.9 mL，P =0.15）。 ANP组（3/57，5%）和安慰剂组（7/60，12%）90天内SAE发生率相似（P =0.36）。血清汞和砷浓度的变化相似。对于大动脉粥样硬化患者，ANP可减少14天时的脑梗死体积（中位数差异：-12.3 mL；四分位差：-27.7 mL至-0.3 mL，P =0.03）。
结论：
ANP 的安全性与安慰剂相似，且在中重度卒中患者中未观察到显著减少脑梗死体积的趋势。未来仍需进一步研究评估 ANP 在减少脑梗死和改善临床预后方面的疗效。
步道登记：
Clinicaltrials.gov，编号NCT04475328。
介绍
最终梗死体积是急性缺血性卒中 (AIS) 患者神经系统预后的有力预测指标。[ 1,2 ]大的梗死体积与不良预后相关，可通过静脉溶栓和血管内治疗 (EVT) 减少梗死体积，即通过血管再通来挽救半暗带。[ 3,4 ]血管重建的时间窗已从 3 小时延长至 24 小时。[ 5,6 ]然而，先前的研究发现，早期梗死的生长持续到卒中后 5 天，[ 7,8 ]并且随着卒中发作和再灌注治疗之间的间隔增加，益处降低。[ 9,10 ]先前的研究试图延长静脉溶栓的时间窗，但不幸的是，没有成功。[ 11,12 ]因此，无论梗死是否生长，卒中发作 4.5 小时后的患者都不允许进行静脉溶栓。临床实践中，超过半数因大血管闭塞引起缺血性卒中患者尽管接受了 EVT，但在随访中仍被发现残疾或死亡。[ 4 ]限制再灌注疗效的一个因素是梗塞生长，即使 EVT 后再灌注充分，梗塞仍会发生，并且预后不良。[ 13,14 ]梗塞生长的潜在机制仍不清楚。年龄、基线梗塞体积、血糖异常、侧支循环、不完全再灌注状态以及开始到再灌注的时间都会影响梗塞生长。[ 13–16 ]此外，EVT 期间每次额外通过都会导致 EVT 后梗塞体积增加 14%。[ 14 ]而且，未接受再灌注治疗的患者梗塞生长的风险更高。[ 17 ]因此，最大限度地减少梗塞生长可能最终会改善临床结果，特别是对于中度至重度卒中患者。
大面积梗塞患者面临的另一项挑战是脑水肿，这是卒中后继发性损伤的已知原因，与高发病率和死亡率相关。[ 18,19 ]减压开颅术可降低恶性脑水肿的死亡率，但发病率也很高。[ 20,21 ] GAMES-RP 研究表明，静脉注射格列苯脲有可能减少大面积半球卒中患者的脑水肿，但并不能改善功能预后。[ 22 ]然而，目前尚无有效药物可预防梗塞生长或脑水肿。
临床前研究结果表明，安宫牛黄丸（ANPs）能够减少梗死体积和脑水肿，并在临床和临床前研究中表现出抗动脉粥样硬化和心脏保护作用。[ 23–25 ]此外，一项随机对照试验的荟萃分析发现，ANP 降低了 AIS 患者的神经功能缺损评分，提高了格拉斯哥昏迷量表，[ 26 ]且不良事件（AE）风险低。[ 27 ]越来越多的证据表明 ANP 是减少梗死生长和脑水肿的候选药物，但其疗效需要在精心设计的临床试验中得到验证。
ANGONG试验是一项先导性研究，旨在探讨ANP对中重度AIS患者的安全性和有效性。基于梗塞生长动力学和目前再灌注治疗的时间窗，我们将ANP的处方时间设定为卒中发作后36小时。我们假设ANP在入组后14天即可安全地减少梗塞生长和脑水肿。
方法
研究设计和参与者
ANGONG 试验是一项在中国 17 家医院开展的随机、双盲、安慰剂对照、多中心临床试验。指导委员会设计并监督了试验和分析。本研究遵循中国《药物临床试验质量管理规范》和《赫尔辛基宣言》进行。本试验经北京协和医院伦理委员会批准（批准号：JS-2371），并获得了所有参与者的书面知情同意。本研究已在 clinicaltrials.gov 注册，注册号为 NCT04475328。
: 所有在发病后 36 小时内到本研究任一中心就诊的 40 至 80 岁连续 AIS 患者均根据纳入和排除标准进行资格筛选。纳入标准为：（1）年龄 40 至 80 岁，（2）诊断为 AIS，（3）诊断为颈内动脉系统急性脑梗死，（4）美国国立卫生研究院卒中量表 (NIHSS) 评分为 10 至 20 分，（5）从症状出现到随机分组的时间为 36 小时内，（6）提供知情同意。如果患者经中医辩证处理后不适合服用 ANP、已接受或计划接受 EVT、AIS 后发生出血性转化或在卒中发作前一个月内接受过 ANP，则将其排除。完整的排除标准见补充材料https://links.lww.com/CM9/B997。
我们成立了一个独立的临床事件裁定委员会，该委员会对试验分组情况不知情，负责评估主要和次要结果。此外，我们还设立了一个数据和安全监察委员会，负责监督试验并定期监测安全事件。
随机化和掩蔽
采用中心分层区组随机化方法，符合条件的参与者按1:1的比例随机分配接受ANP或安慰剂治疗（3克/片，每日1片，共5天）。患者根据入组时间被分配随机序列号，并被提供相应的药物，这些药物事先已设盲。主要研究者、研究中心研究者、医生、患者和结果工作人员均对治疗方案不知情。两组的药片颜色、形状和包装均相同。
程序
临床程序
参与者随机分组后，立即接受口服或鼻腔给药。给药方案基于临床实践和既往ANP临床研究。[ 26 ]合并治疗按照中华医学会神经病学分会的指南进行。[ 28 ]根据标准指南，抗血小板治疗和他汀类药物作为标准治疗。[ 28 ]
基线收集人口统计学数据、症状和体征、病史、实验室检查结果、美国国立卫生研究院卒中量表（NIHSS）评分和改良Rankin量表（mRS）评分。由训练有素的资深神经科医生在第14天和第90天通过现场访视或电话方式使用标准化问卷评估NIHSS和mRS评分。基线、第7天和第14天分别进行格拉斯哥昏迷量表（GCS）评估。基线和第7天进行心电图（ECG）检查。基线、第2天和第7天采集血液样本。为了检测汞和砷的浓度，将第2天和第7天的血液样本离心并储存在-20℃下进行集中分析。
图像处理
在研究入组和随机分组前，研究人员进行了脑部 MRI (3.0T) 检查，包括 T1W、弥散加权成像 (DWI)、表观弥散系数 (ADC) 和液体衰减反转恢复 (FLAIR) 序列。后续随访于第 14 天和第 90 天进行脑部磁共振成像 (MRI)，使用与基线时相同的 MRI 设备。成像参数列于补充表 1，https://links.lww.com/CM9/B997。
所有图像均使用AccuBrain2.0®（深圳脑诺医疗科技有限公司）集中处理。AccuBrain2.0®可以自动分割和量化大脑结构和区域，并且精度很高。[ 29 ]根据 T1W MRI 数据，经验丰富的放射科医生自动分割出包括海马、侧脑室和杏仁核在内的大脑结构，以及三种主要脑组织（白质、灰质和脑脊液）。记录了大脑结构和区域的绝对体积（mL）和相对体积（以颅内容量 [ICV] 标准化的百分比）。用“萎缩率”测量皮质区域，萎缩率定义为每个叶中脑脊液（CSF）体积与实质体积的比率。在对每个受试者的 FLAIR 和 DWI MRI 扫描结果进行比对后，由训练有素的专家以半自动模式分割脑梗死。 T2-FLAIR 和 DWI MRI 图像上均一致的高信号区域被识别为梗死。本研究中，脑梗死是通过 DWI 图像检测的。脑水肿定义为 T2-FLAIR 图像上呈高信号，但 DWI 图像上无高信号的区域。记录脑梗死和脑水肿的绝对体积（mL）。
结果
主要疗效结局指标为治疗14天后脑梗死及脑水肿体积的变化。次要疗效结局指标为：(1) 90天后脑梗死及脑水肿体积的变化；(2) 14天和90天后NIHSS评分的变化；(3) 14天和90天时mRS评分为0～2分的患者比例；(4) 7天和14天后GCS评分的变化。
主要安全性结局指标为90天内发生严重不良事件（SAE）的比例，其他安全性结局指标包括：（1）7天内SAE的比例；（2）7天后血常规、生化检查及血汞、砷浓度的变化；（3）7天后心电图的变化；（4）7天和90天内AE的比例；（5）7天和90天内全因死亡的比例；（6）7天和90天内合并血管事件（缺血性卒中、出血性卒中、心肌梗死或血管性死亡）的比例。SAE分为：（1）死亡；（2）危及生命的事件；（3）住院（初次或长期）；（4）严重或持续性残疾/损伤；（5）先天性异常/出生缺陷；（6）研究者确定的其他严重医学事件。
统计分析
在研究开始之前，我们根据 ANP 会减少随访中的脑梗死和水肿体积的假设设定了样本量。[ 25,26 ]在大脑中动脉闭塞的小鼠中，ANP 使梗死体积比例从 20% 降低到 2%。[ 25 ]由于缺乏关于 ANP 预防人类脑梗死增长的数据，该研究旨在连续招募 120 名符合条件的中度至重度 AIS 参与者。
基于意向治疗分析 (ITT)，我们建立了全分析集 (FAS)。该分析集包含已随机分组并接受研究药物治疗的参与者。因此，我们在分析中采用了改良的 ITT 方法。
FAS 是用于疗效分析的主要人群。符合方案集 (PPS) 包括所有完成方案中规定治疗或未严重违反方案的参与者。PPS 是用于评估疗效的次要分析人群。安全性集 (SS) 包括至少接受过一次治疗的参与者。基线数据根据治疗分配情况呈现，并酌情使用描述性统计信息。
各治疗组间主要疗效指标的疗效分析采用Wilcoxon秩和检验，以FAS和PPS为样本，比较各治疗组间疗效中位数变化，并采用Hodges-Lehmann法计算中位数差（MD）和置信区间（CI）。次要疗效指标NIHSS和GCS评分变化也采用类似方法进行分析。采用逻辑回归分析，将试验中心设定为随机效应，报告mRS评分0-2的比值比（OR）和95%置信区间。构建Kaplan-Meier曲线，比较各治疗组90天全因死亡率。此外，我们还评估了ANP在预设亚组中的疗效。
大动脉粥样硬化（LAA）被认为是中度至重度卒中的主要原因，因此对诊断为 LAA 的患者进行了主要和次要结果的敏感性分析，从而为进一步评估 ANP 的疗效提供线索。
所有安全性分析均针对SS进行，并以各治疗组安全性事件的发生频率和百分比表示。采用Wilcoxon秩和检验（用于血液检查结果以及汞和砷浓度的变化）、χ2检验或Fisher精确概率法比较各治疗组。
在主要疗效分析中删除缺失值。所有检验均为双侧检验，P值<0.05被认为具有统计学意义。所有统计分析均使用SAS软件（版本9.4；SAS Institute，美国北卡罗来纳州卡里）进行。
结果
2021年4月至2022年7月期间，共入组120名患者。ANP组和安慰剂组各60名参与者。ANP组中，3名患者未接受研究药物，而安慰剂组中，60名患者接受了研究药物。因此，117名参与者保留在FAS中进行意向治疗分析。ANP组和安慰剂组中分别有9名和8名患者未接受主要结果评估。此外，安慰剂组中有3名患者在随机分组后不符合纳入标准，ANP组中有1名患者接受了试验禁用药物。最终，96名参与者被纳入符合方案分析[图1 ]。FAS组和非FAS组的基线年龄、性别、高血压、糖尿病和NIHSS评分均具有可比性[补充表2，https://links.lww.com/CM9/B997 ]。两组患者的基线人口统计学、临床和放射学特征以及实验室检查结果相似 [表 1 ]。患者年龄中位数为 66.0 岁（四分位距 [IQR]：58.0–73.0 岁），34%（40/117）为女性。NIHSS 评分中位数为 12.0（IQR：11.0–15.0）。意向治疗分析显示，ANP 组从症状出现到给药的中位时间为 27 小时，安慰剂组为 27 小时 [表 1 ]。两组患者症状出现后静脉注射重组组织型纤溶酶原激活剂 (rtPA) 的比例相似（25% [14/57] vs 23% [14/60]）。 ANP 组和安慰剂组的伴随治疗相当 [补充表 3，https://links.lww.com/CM9/B997 ]。
Background:
Preclinical studies have indicated that Angong Niuhuang Pills (ANP) reduce cerebral infarct and edema volumes. This study aimed to investigate whether ANP safely reduces cerebral infarct and edema volumes in patients with moderate to severe acute ischemic stroke.
Methods:
This randomized, double-blind, placebo-controlled pilot trial included patients with acute ischemic stroke with National Institutes of Health Stroke Scale (NIHSS) scores ranging from 10 to 20 in 17 centers in China between April 2021 and July 2022. Patients were allocated within 36 h after onset via block randomization to receive ANP or placebo (3 g/day for 5 days). The primary outcomes were changes in cerebral infarct and edema volumes after 14 days of treatment. The primary safety outcome was severe adverse events (SAEs) for 90 days.
Results:
There were 57 and 60 patients finally included in the ANP and placebo groups, respectively for modified intention-to-treat analysis. The median age was 66.0 years, and the median NIHSS score at baseline was 12.0. The changes in cerebral infarct volume at day 14 were 0.3 mL and 0.4 mL in the ANP and placebo groups, respectively (median difference: –7.1 mL; interquartile range [IQR]: −18.3 to 2.3 mL, P = 0.30). The changes in cerebral edema volume of the ANP and placebo groups on day 14 were 11.4 mL and 4.0 mL, respectively (median difference: 3.0 mL, IQR: −1.3 to 9.9 mL, P = 0.15). The rates of SAE within 90 days were similar in the ANP (3/57, 5%) and placebo (7/60, 12%) groups (P = 0.36). Changes in serum mercury and arsenic concentrations were comparable. In patients with large artery atherosclerosis, ANP reduced the cerebral infarct volume at 14 days (median difference: –12.3 mL; IQR: –27.7 to –0.3 mL, P = 0.03).
Conclusions:
ANP showed a similar safety profile to placebo and non-significant tendency to reduce cerebral infarct volume in patients with moderate-to-severe stroke. Further studies are warranted to assess the efficacy of ANP in reducing cerebral infarcts and improving clinical prognosis.
Trail Registration:
Clinicaltrials.gov, No. NCT04475328.
Introduction
Final infarct volume is a strong predictor of neurological outcomes in patients with acute ischemic stroke (AIS).[1,2] A large infarct volume, which is associated with poor outcomes, can be reduced by intravenous thrombolysis and endovascular therapy (EVT), which salvages the penumbra via vessel recanalization.[3,4] The time window for revascularization has been extended from 3 h to 24 h.[5,6] However, previous studies found that early infarct growth continued up to 5 days after stroke,[7,8] and the benefit decreased as the interval between the time of stroke onset and reperfusion therapy grew.[9,10] Previous studies attempted to extend the time window of intravenous thrombolysis, but unfortunately, without success.[11,12] Thus, intravenous thrombolysis is not permitted in patients after 4.5 h of stroke onset, regardless of infarct growth. In clinical practice, over half of the patients with ischemic stroke caused by large-vessel occlusions were found to be disabled or dead at follow-up, despite undergoing EVT.[4] One factor limiting the efficacy of reperfusion is infarct growth, which occurs despite adequate reperfusion after EVT and predicts a poor outcome.[13,14] The underlying mechanism of infarct growth remains unclear. Age, baseline infarct volume, dysglycemia, collateral circulation, incomplete reperfusion status, and onset-to-reperfusion time can influence infarct growth.[13–16] In addition, each extra pass during EVT leads to a 14% increase in post-EVT infarct volume.[14] Moreover, patients without reperfusion therapy have a higher risk of infarct growth.[17] Thus, minimizing infarct growth might ultimately improve clinical outcomes, especially in patients with moderate-to-severe stroke.
Another challenge in patients with a large infarct is cerebral edema, a known cause of secondary injury after stroke onset, which is associated with high morbidity and mortality.[18,19] Decompressive craniectomy can reduce mortality from malignant cerebral edema but shows substantial morbidity.[20,21] The GAMES-RP study revealed that intravenous glyburide has the potential to reduce cerebral edema in patients with large hemispheric strokes but did not improve the functional prognosis.[22] However, no effective drug is currently available to prevent infarct growth or cerebral edema.
Results of preclinical studies suggested that Angong Niuhuang Pills (ANPs), a traditional Chinese patent medicine, including realgar, cinnabaris, Bovis Calculus, artificial Moschus, and powdered buffalo horn extract, decreased infarct volume and cerebral edema, and presented anti-atherosclerosis and cardio-protective effects in clinical and preclinical studies.[23–25] Furthermore, a meta-analysis of randomized controlled trials found that ANP reduced the neurologic deficit score and increased the Glasgow Coma Scale in patients with AIS,[26] with a low risk of adverse events (AEs).[27] Increasing evidence indicates that ANP is a candidate drug for reducing infarct growth and cerebral edema, but its efficacy needs to be verified in a well-designed clinical trial.
The ANGONG TRIAL was designed as a pilot study to explore the safety and efficacy of ANP in patients with moderate-to-severe AIS. Based on infarct growth dynamics and the current time window for reperfusion therapy, we set the time of ANP prescription to 36 h after stroke onset. We hypothesized that ANP would safely reduce infarct growth and cerebral edema 14 days after enrollment.
Methods
Study design and participants
The ANGONG TRIAL was a randomized, double-blind, placebo-controlled, multicenter trial conducted in 17 Chinese hospitals. The steering committee designed and supervised the trial and analysis. This study was conducted in accordance with the Guidelines for Good Clinical Practice in China and the Declaration of Helsinki. This trial was approved by the Ethics Committee of Peking Union Medical College Hospital (No. JS-2371), and written informed consent was obtained from all participants. The study was registered at clinicaltrials.gov, No. NCT04475328.
All consecutive patients with AIS aged 40–80 years visiting any center in our study within 36 h from onset were screened for eligibility according to the inclusion and exclusion criteria. The inclusion criteria were: (1) an age of 40–80 years, (2) diagnosis of AIS, (3) diagnosis of acute cerebral infarction of the internal carotid artery system, (4) a National Institutes of Health Stroke Scale (NIHSS) score ranging from 10 to 20, (5) a time from symptom onset to randomization within 36 h, and (6) provision of informed consent. Patients were excluded if they were found not suitable for taking ANP after the dialectical process by a traditional Chinese medical doctor, received or planned to receive EVT, if they had hemorrhagic transformation after AIS, or received ANP within one month before stroke onset. The full exclusion criteria are presented in Supplementary Material, https://links.lww.com/CM9/B997.
An independent clinical event adjudication committee masked to the trial group assignments was established to assess the primary and secondary outcomes. In addition, a data and safety monitoring board oversaw the trial and regularly monitored safety events.
Randomization and masking
Eligible participants were randomly assigned (1:1) to receive either ANP or placebo (3 g/pill, 1 pill/day for 5 days) using a central stratified block randomization method. Patients were assigned random serial numbers based on their time of enrolment and were provided with the corresponding medicines, which were blind-covered beforehand. The principal investigators, site investigators, physicians, patients, and outcome personnel were blinded to the treatment. For both groups, the pill colors, shapes, and packaging were identical.
Procedures
Clinical procedures
Treatments were orally or nasally administered to the participants immediately after randomization. The dosing schedule was based on clinical practice and previous clinical studies of ANP.[26] Concomitant treatments were administered according to the guidelines from the Chinese Society of Neurology.[28] Antiplatelet therapy and statins were used as a standard of care based on standard guidelines.[28]
Demographic data, signs and symptoms, medical history, laboratory test results, NIHSS scores, and modified Rankin Scale (mRS) scores were collected at baseline. The NIHSS and mRS scores were assessed on days 14 and 90 by a trained senior neurologist using a standardized questionnaire via site visits or telephone. The Glasgow Coma Scale (GCS) was assessed at baseline and on days 7 and 14. Electrocardiography (ECG) was performed at baseline and on day 7. Blood samples were obtained at baseline and on days 2 and 7. To detect the concentrations of mercury and arsenic, blood samples on days 2 and 7 were centrifuged and stored at −20°C for centralized analysis.
Image procedures
Brain MRI (3.0T), including T1W, diffusion weighted imaging (DWI), apparent diffusion coefficient (ADC), and fluid attenuated inversion recovery (FLAIR) series, was performed prior to study enrollment and randomization. Follow-up brain magnetic resonance imagings (MRIs) were conducted on days 14 and 90 with the same MRI machine used at baseline. The imaging parameters are listed in Supplementary Table 1, https://links.lww.com/CM9/B997.
All images were centrally processed using AccuBrain2.0® (Brainnow Medical Technology Ltd., Shenzhen, China). AccuBrain2.0® can automatically segment and quantify brain structures and regions with high accuracy.[29] Given the T1W MRI data, brain structures including the hippocampus, lateral ventricle, and amygdala, with three major brain tissues (white matter, grey matter, and cerebrospinal fluid) were automatically segmented by experienced radiologists. The absolute (mL) and relative volumes (% normalized by intracranial volume [ICV]) of the brain structures and regions were recorded. The cortical regions were measured with “atrophy ratios”, which were defined as the ratio of the volume of cerebrospinal fluid (CSF) to the volume of parenchyma in each lobe. After aligning each subject’s FLAIR and DWI MRI scans, brain infarcts were segmented by a trained expert in semi-automatic mode. Hyperintensities with correspondences in both T2-FLAIR and DWI MRI were identified as infarcts. In our study, cerebral infarctions were detected using DWI. Cerebral edema was defined as a region with hyperintensity on T2-FLAIR but not on DWI. The absolute volumes (mL) of the cerebral infarct and cerebral edema were recorded.
Outcomes
The primary outcomes were changes in cerebral infarct and edema volumes that changed after 14 days of treatment. The secondary efficacy outcomes were (1) changes in cerebral infarct and cerebral edema volume after 90 days, (2) changes in the NIHSS score after 14 days and 90 days, (3) the proportion of patients with an mRS score of 0–2 at days 14 and 90; and (4) changes in GCS after 7 days and 14 days.
The primary safety outcome was the proportion of severe adverse event (SAE) occurred within 90 days. The other safety outcomes included (1) the proportion of SAE within 7 days; (2) changes in routine blood examination, biochemical test, and mercury and arsenic concentrations after 7 days; (3) changes in electrocardiogram after 7 days; (4) proportions of AEs within days 7 and 90; (5) proportions of all-cause mortality within 7 days and 90 days; (6) proportions of combined vascular events (ischemic stroke, hemorrhagic stroke, myocardial infarction, or vascular death) within 7 days and 90 days. SAE was classified into (1) death, (2) life-threatening events, (3) hospitalization (initial or prolonged), (4) significant or persistent disability/damage, (5) congenital anomaly/birth defect, and (6) other serious medical events determined by the investigator.
Statistical analysis
Before the start of the study, the sample size was set based on our hypothesis that ANP would reduce the cerebral infarct and edema volume at follow-up.[25,26] ANP decreased the infarct volume ratio from 20% to 2% in mice with middle cerebral artery occlusion.[25] Due to the lack of data on ANP regarding the prevention of cerebral infarct growth in humans, the study was designed to consecutively enroll 120 eligible participants with moderate-to-severe AIS.
A full analysis set (FAS) was established based on intention-to-treat analysis (ITT). This set included participants who had undergone randomization and received the study drug. Thus, modified ITT was adopted in our analysis.
The FAS was the primary population used for efficacy analysis. The per-protocol set (PPS) included all participants who completed the treatment specified in the protocol or who did not seriously violate the protocol. The PPS was the secondary analysis population used to assess efficacy. The safety set (SS) included participants who received at least one dose of the treatment. Baseline data were presented according to treatment assignment, with descriptive statistics as appropriate.
Efficacy analysis of the primary outcome between treatment groups was conducted on the FAS and PPS using the Wilcoxon rank-sum test to compare the median change in response across treatment groups, and the Hodges–Lehmann method was used to calculate the median difference (MD) and confidence intervals (CIs). A similar approach was used for the secondary outcomes of changes in NIHSS and GCS scores. Logistic regression, with the trial centers set as a random effect, odds ratios (ORs), and 95% CIs were reported for mRS scores of 0–2. A Kaplan–Meier curve was constructed to compare the treatment groups for all-cause mortality at 90 days. Moreover, we assessed the efficacy of ANP in pre-specified subgroups.
As large artery atherosclerosis (LAA) is thought to be the main cause of moderate-to-severe stroke, a sensitivity analysis of the primary and secondary outcomes was conducted in patients diagnosed with LAA, thus providing clues for further efficacy assessment of ANP.
All safety analyses were conducted on SS and are presented as frequencies and percentages of safety events by treatment group. The Wilcoxon rank-sum test (for changes in blood tests and concentrations of mercury and arsenic), χ2 test, or Fisher’s exact test was used to compare treatment groups.
Missing values were deleted in the primary efficacy analysis. All tests were two-sided, and P-values <0.05 were considered statistically significance. All statistical analyses were conducted using the SAS software (version 9.4; SAS Institute, Cary, North Carolina, U.S.).
Results
Between April 2021 and July 2022, a total of 120 patients were enrolled. Sixty participants were randomly assigned to each of the ANP and placebo groups. In the ANP group, three patients did not receive the study drug, while in the placebo group, 60 patients received the study drug. Thus, 117 participants remained in the FAS for the intention-to-treat analysis. Nine and eight additional patients failed to undergo primary outcome assessment in ANP group and placebo group, respectively. Moreover, three patients in the placebo group did not meet the inclusion criteria after randomization, and one patient in the ANP group received trial-prohibited drugs. Finally, 96 were included in the per-protocol analysis [Figure 1]. Age, sex, hypertension, diabetes mellitus, and NIHSS score at baseline were comparable between FAS and non-FAS groups [Supplementary Table 2, https://links.lww.com/CM9/B997]. Demographic, clinical, and radiological characteristics and laboratory test results at baseline were comparable between the two treatment groups [Table 1]. The median age of the patients was 66.0 years (interquartile range [IQR]: 58.0–73.0 years), and 34% (40/117) were female. The median NIHSS score was 12.0 (IQR: 11.0–15.0). In the intention-to-treat analysis, the median time from symptom onset to drug administration was 27 h in the ANP group and 27 h in the placebo group [Table 1]. The rates of intravenous recombinant tissue plasminogen activator (rtPA) administration after symptom onset were similar between the two groups (25% [14/57] vs. 23% [14/60]). Concomitant treatments were comparable between the ANP and placebo groups [Supplementary Table 3, https://links.lww.com/CM9/B997].
Flow chart of ANGONG TRIAL. ANGONG TRIAL: Safety and efficacy of Angong Niuhuang Pills in patients with moderate-to-severe acute ischemic stroke; ANP: Angong Niuhuang Pills; MRI: Magnetic resonance imaging; NIHSS: National Institutes of Health Stroke Scale.
Table 1 - Demographic, clinical, and radiological characteristics of ANGONG TRIAL.
| Variables | ANP (n = 57) | Placebo (n = 60) | Statistical values | P values |
|---|---|---|---|---|
| Age (years) | 66.0 (57.0–72.0) | 66.0 (58.0–74.0) | 0.14 | 0.71 |
| Female | 19 (33) | 21 (35) | 0.04 | 0.85 |
| Ethnic minorities | 1 (2) | 1 (2) | 0.00 | 0.97 |
| Education, middle school and above | 18 (32) | 13 (22) | 1.47 | 0.22 |
| Medical history | ||||
| Hypertension | 34 (60) | 37 (62) | 0.05 | 0.82 |
| Diabetes mellitus | 9 (16) | 14 (23) | 1.05 | 0.30 |
| Hyperlipidaemia | 3 (5) | 5 (8) | – | 0.72 |
| Ischaemic stroke | 15 (26) | 13 (22) | 0.35 | 0.56 |
| Transient anemia attack | 2 (4) | 2 (3) | – | >0.99 |
| Cerebral hemorrhage | 1 (2) | 0 | – | 0.49 |
| Coronary artery disease | 6 (11) | 4 (7) | – | 0.52 |
| Atrial fibrillation | 9 (16) | 9 (15) | 0.01 | 0.91 |
| Smoking, ever | 22 (39) | 22 (37) | 0.05 | 0.83 |
| Drinking, ever | 20 (35) | 20 (33) | 0.04 | 0.84 |
| Family history of stroke | 1 (2) | 3 (5) | 0.93 | 0.33 |
| Past medication history | ||||
| Antiplatelet agent | 10 (18) | 14 (23) | 0.60 | 0.44 |
| Anticoagulation agent | 2 (4) | 3 (5) | – | >0.99 |
| Lipid-lowering agent | 9 (16) | 13 (22) | 0.66 | 0.42 |
| Antihypertensive agent | 23 (40) | 23 (38) | 0.05 | 0.82 |
| Antidiabetic agent | 6 (11) | 11 (18) | 1.43 | 0.23 |
| Agent containing ingredient of ANP | 2 (4) | 1 (2) | – | >0.99 |
| Body mass index at baseline (kg/m2) | 24.3 (22.1–26.1) | 24.2 (22.2–26.6) | 0.01 | 0.93 |
| Baseline systolic blood pressure (mmHg) | 153 (136–166) | 148 (134–166) | 0.34 | 0.56 |
| Baseline diastolic blood pressure (mmHg) | 86 (80– 99) | 86 (78–95) | 0.55 | 0.46 |
| NIHSS score at baseline | 13 (11– 15) | 12 (10–15) | 1.61 | 0.20 |
| Glasgow coma scale at baseline | 13 (11– 14) | 13 (11–15) | 0.63 | 0.43 |
| mRS score before symptom onset | – | 0.67 | ||
| 0 | 54 (95) | 58 (97) | ||
| 1 | 3 (5) | 2 (3) | ||
| Baseline DWI infarction volume (mL) | 41.4 (12.2–101.7)* | 32.1 (9.5–88.8) | 0.74 | 0.39 |
| Baseline cerebral edema volume (mL) | 7.4 (3.9–16.1)† | 4.3 (1.8–15.5) | 0.99 | 0.32 |
| TOAST classification‡ | – | 0.72 | ||
| Large artery atherosclerosis | 36 (67) | 40 (69) | ||
| Cardioembolism | 8 (15) | 9 (16) | ||
| Small artery | 8 (15) | 8 (14) | ||
| Other | 2 (4) | 0 (0) | ||
| Unknown | 0 (0) | 1 (2) | ||
| Intravenous rtPA after onset | 14 (25) | 14 (23) | 0.02 | 0.88 |
| Laboratory test | ||||
| White blood cell (109/L) | 7.9 (6.5–9.2)§ | 8.0 (7.1–9.7) | 0.24 | 0.63 |
| Neutrophil (109/L) | 5.8 (4.8–7.1)§ | 6.3 (4.6–8.0) | 0.43 | 0.51 |
| Hemoglobin (g/L) | 140 (133– 152)§ | 145 (129–150) | 0.00 | 0.99 |
| Platelet (109/L) | 211 (175– 255)§ | 199 (166– 249) | 0.86 | 0.35 |
| Alanine transaminase (U/L) | 17 (13– 22)|| | 18 (13–25) | 0.33 | 0.56 |
| Creatinine (mmol/L) | 70 (56–76)¶ | 69 (59–89)** | 2.14 | 0.14 |
| Fasting blood glucose (mmol/L) | 6.0 (5.4–7.7)§ | 6.6 (5.7–7.3) | 0.20 | 0.66 |
| Na (mmol/L) | 140 (138–142) | 140 (138–142)†† | 0.17 | 0.68 |
| PT (s) | 12 (11–13)‡‡ | 12 (101–13)§§ | 0.50 | 0.48 |
| APTT (s) | 27 (26–30)|||| | 28 (25–30)¶¶ | 0.03 | 0.87 |
| LDL-C (mmol/L) | 2.9 (2.3–3.6)*** | 2.7 (2.3–3.5)††† | 0.14 | 0.71 |
| High sensitivity C reactive protein (mg/L) | 4.9 (2.2–17.0) | 6.1 (2.7–11.3) | 0.01 | 0.94 |
| Symptom onset to study drug (h) | 27 (18–33) | 27 (21–33) | 0.96 | 0.33 |
| Symptom onset to baseline MRI (h) | 24(12–30) | 24 (12– 33) | 0.39 | 0.53 |
Data are presented as median (IQR) or n (%). Statistical values for data shown as n (%) are χ2; for data shown as median (IQR) are Z values; – for not applicable. *56 patients were analyzed. †55 patients were analyzed. ‡54 patients were analyzed. §56 patients were analyzed. ||55 patients were analyzed. ¶57 patients were analyzed. **59 patients were analyzed. ††59 patients were analyzed. ‡‡53 patients were analyzed. §§59 patients were analyzed. ||||53 patients were analyzed. ¶¶58 patients were analyzed. ***55 patients were analyzed. †††57 patients were analyzed. ANGONG TRIAL: Safety and efficacy of Angong Niuhuang Pills in patients with moderate-to-severe acute ischemic stroke; ANP: Angong Niuhuang Pill; APTT: Activated partial thromboplastin time; DWI: Diffusion weighted imaging; GCS: Glasgow coma scale; IQR: Interquartile range; LAA: Large artery atherosclerosis; LDL-C: Low-density lipoprotein cholesterol; MRI: Magnetic resonance imaging; mRS: Modified rankin scale; NIHSS: National Institutes of Health Stroke Scale; PT: Prothrombin time; rtPA: Recombinant tissue plasminogen activator; TOAST: Trial of Org 10172 in Acute Stroke Treatment.
In the intention-to-treat analysis, the changes in cerebral infarct volume at 14 days were 0.3 mL in the ANP group and 0.4 mL in the placebo group (MD: −7.1 mL, IQR: −18.3 to 2.3 mL, P = 0.30). The changes in cerebral edema volume of ANP and placebo groups were 11.4 mL and 4.0 mL, respectively (MD: 3.0 mL, IQR: −1.3 to 9.9 mL, P = 0.15). There were no differences in secondary efficacy outcomes between the ANP and placebo groups [Table 2].
Table 2 - Efficacy outcomes based on intention-to-treat analysis of ANGONG TRIAL.
| Variables | ANP (n = 57) | Placebo (n = 60) | Measurement of effect size | Effect size (95% CI) | P values |
|---|---|---|---|---|---|
| Primary outcome | |||||
| Change in cerebral infarction volume at 14 days (mL) | 0.3 (–17.4 to 7.6)* | 0.4 (–7.0 to 16.2)† | MD | –7.1 (–18.3 to 2.3) | 0.30 |
| Change in cerebral edema volume at 14 days (mL) | 11.4 (1.3–23.7)‡ | 4.0 (0.4–18.5)§ | MD | 3.0 (–1.3 to 9.9) | 0.15 |
| Secondary outcome | |||||
| Change in cerebral infarction volume at 90 days (mL) | –5.3 (–32.7 to 2.6)|| | –2.3 (–19.0 to 10.4)¶ | MD | 3.9 (–5.5 to 23.1) | 0.54 |
| Change in cerebral edema volume at 90 days (mL) | 2.7 (–0.9 to 17.1)** | 3.1 (0.4–14.1)†† | MD | 0.6 (–3.9 to 4.7) | 0.89 |
| mRS 0–2 at 14 days | 7/54 (13) | 10/57 (18) | OR | 0.7 (0.3–2.0) | 0.50 |
| mRS 0–2 at 90 days | 17/45 (38) | 12/49 (24) | OR | 1.9 (0.8–4.6) | 0.16 |
| Change in NIHSS score at 14 days | –2.5 (–5.0 to –1.0)‡‡ | –3.0 (–6.0 to –1.0)§§ | MD | 0 (–1.0 to 1.0) | 0.91 |
| Change in NIHSS score at 90 days | –6.0 (–9.0 to –4.0)|||| | –6.0 (–7.0 to –4.0)¶¶ | MD | –1.0 (–2.0 to 1.0) | 0.46 |
| Change in Glasgow coma score at 7 days | 0 (0–1.0)*** | 0 (0–1.0)††† | MD | 0 | 0.05 |
| Change in Glasgow coma score at 14 days | 0 (0–1.0)‡‡‡ | 0 (0–1.0)§§§ | MD | 0 (0–1.0) | 0.05 |
Data were presented as median (IQR) or n (%). *48 patients were analyzed. †52 patients were analyzed. ‡48 patients were analyzed. §51 patients were analyzed. ||38 patients were analyzed. ¶35 patients were analyzed. **37 patients were analyzed. ††35 patients were analyzed. ‡‡54 patients were analyzed. §§57 patients were analyzed. ||||45 patients were analyzed. ¶¶48 patients were analyzed. ***54 patients were analyzed. †††58 patients were analyzed. ‡‡‡54 patients were analyzed. §§§57 patients were analyzed. ANGONG TRIAL: Safety and efficacy of Angong Niuhuang Pills in patients with moderate-to- severe acute ischemic stroke; ANP: Angong Niuhuang Pill; CI: Confidence interval; GCS: Glasgow coma scale; IQR: Interquartile range; MD: Median difference; mRS: Modified rankin scale; NIHSS: National Institutes of Health Stroke Scale; OR: Odds ratio.
The per-protocol analysis included 96 participants, 47 of whom were allocated to the ANP group and 49 to the placebo group. The change in cerebral infarct volume in the ANP group was similar to that in the placebo group (0.3 mL vs. 0.7 mL, MD: −8.2 mL, IQR: –20.3 to 2.3 mL, P = 0.18). In addition, the change in cerebral edema volume at day 14 was 9.3 mL in the ANP group and 4.4 mL in the placebo group; however, the difference was not statistically significant (2.6 [–1.8 to 9.6], P = 0.25). A mRS score of 0–2 was achieved in 22% (9/41) of the patients in the placebo group and 38% (15/40) in the ANP group (OR: 2.1, 95% CI: [0.8–5.7], P = 0.13). Other secondary outcomes were comparable between the two treatment groups [Supplementary Table 4, https://links.lww.com/CM9/B997].
Subgroup analysis of the primary outcomes in the FAS showed that ANP significantly reduced infarct growth in ischemic stroke with LAA [Supplementary Table 5, https://links.lww.com/CM9/B997]. To further assess the efficacy of ANP, we conducted sensitivity analyses in patients with LAA. In FAS, the change in cerebral infarct volume at 14 days was −1.0 mL in the ANP group and 1.2 mL in the placebo group (MD: −12.3 mL, IQR: −27.7 to −0.3 mL; P = 0.03), as shown in Table 3. The per-protocol analysis also showed that the change in cerebral infarct volume at day 14 in the ANP group was significantly lower than that in the placebo group (MD: −13.6 mL, IQR: −29.8 to −0.4 mL), with a P-value of 0.04 [Supplementary Table 6, https://links.lww.com/CM9/B997]. There were no differences in the changes of cerebral edema volume or secondary efficacy outcomes between the ANP and placebo groups.
Table 3 - Sensitivity analysis in patients with LAA based on intention-to-treat analysis.
| Variables | ANP (n = 36) | Placebo (n = 40) | Measurement of effect size | Effect size (95% CI) | P values |
|---|---|---|---|---|---|
| Primary outcome | |||||
| Change in cerebral infarction volume at 14 days (mL) | –1.0 (–19.4 to 8.3)* | 1.2 (–5.2 to 21.2)† | MD | –12.3 (–27.7 to –0.3) | 0.03 |
| Change in cerebral edema volume at 14 days (mL) | 9.3 (1.3 to 23.6)‡ | 3.8 (–0.4 to 16.5)§ | MD | 4.8 (–1.1 to 11.5) | 0.10 |
| Secondary outcome | |||||
| Change in cerebral infarction volume at 90 days (mL) | –9.0 (–56.5 to 2.6)|| | –3.9 (–15.4 to 10.4)¶ | MD | –14.3 (–40.1 to 3.9) | 0.09 |
| Change in cerebral edema volume at 90 days (mL) | 7.0 (–0.5 to 18.6)** | 1.1 (0.1–10.2)†† | MD | 3.0 (–3.3 to 13.9) | 0.10 |
| mRS 0–2 at 14 days | 5/36 (14) | 7/39 (18) | OR | 0.7 (0.2–2.6) | 0.63 |
| mRS 0–2 at 90 days | 11/28 (39) | 8/33 (24) | OR | 2.0 (0.7–6.1) | 0.21 |
| Change in NIHSS score at 14 days | –2.0 (–4.0 to –1.00) | –3.0 (–6.0 to 0)‡‡ | MD | 0 (–1.0 to 2.0) | 0.60 |
| Change in NIHSS score at 90 days | –6.0 (–9.0 to –3.5)§§ | –5.5 (–7.0 to –4.0)|||| | MD | –1.0 (–2.0 to 1.0) | 0.63 |
| Change in Glasgow coma score at 7 days | 0 (0–1.0) | 0 (0–1.0) | MD | 0 | 0.18 |
| Change in Glasgow coma score at 14 days | 0 (0–1.0) | 0 (0–1.0)¶¶ | MD | 0 (0–1.0) | 0.09 |
Data were presented as median (IQR) or n (%). *31 patients were analyzed. †37 patients were analyzed. ‡31 patients were analyzed. §36 patients were analyzed. ||23 patients were analyzed. ¶25 patients were analyzed. **22 patients were analyzed. ††25 patients were analyzed. ‡‡39 patients were analyzed. §§28 patients were analyzed. ||||32 patients were analyzed. ¶¶39 patients were analyzed. ANP: Angong Niuhuang Pill; CI: Confidence interval; GCS: Glasgow coma scale; IQR: Interquartile range; LAA: Large artery atherosclerosis; MD: Median difference; mRS: Modified rankin scale; NIHSS: National Institutes of Health Stroke Scale; OR: Odds ratio.
The rate of SAE at 90 days was similar between the ANP and placebo groups (5% [3/57] vs. 12% [7/60], respectively; P = 0.36) [Table 4]. By 90 days, 3 (5%) of 57 patients died in the ANP group compared to 2 (3%) of 60 patients in the placebo group (P = 0.95). The Kaplan–Meier curve for all-cause mortality did not indicate a significant difference between the ANP and placebo groups [Supplementary Figure 1, https://links.lww.com/CM9/B997]. In addition, the changes in the concentrations of mercury, arsenic and biochemical indicators on day 7 were similar between the two treatment groups [Table 4 and Supplementary Table 7, https://links.lww.com/CM9/B997]. There were no significant differences in the daily body temperature within 7 days of enrollment between the ANP and placebo groups (data not shown).
Table 4 - Safety outcomes of ANGONG TRIAL.
| Variables | ANP (n = 57) | Placebo (n = 60) | Statistical values | P values |
|---|---|---|---|---|
| SAE up to day 90 | 3 (5) | 7 (12) | 0.82 | 0.36 |
| SAE up to day 7 | 0 | 4 (7) | 2.17 | 0.14 |
| AE up to day 90 | 14 (25) | 12 (20) | 0.35 | 0.55 |
| AE up to day 7 | 10 (18) | 7 (12) | 0.81 | 0.36 |
| All-cause deaths up to day 90 | 3 (5) | 2 (3) | 0 | 0.95 |
| All-cause deaths up to day 7 | 0 | 0 | – | – |
| Combined vascular events to day 90 | 1 (2) | 3 (5) | 0.21 | 0.65 |
| Combined vascular events to day 7 | 0 | 1 (2) | – | >0.99 |
| Abnormal ECGs after baseline | 26 (55) | 25 (58) | 0.07 | 0.79 |
| Change in concentration of mercury (mg/L) | 0* | 0† | 1.31 | 0.25 |
| Change in concentration of arsenic (mg/L) | 0 (–1.1 to 0.3)‡ | 0 (–0.7 to 1.0)§ | 0.01 | 0.94 |
Data were presented as median (IQR) or n (%); statistical values for data shown as n (%) are χ2; for data shown as median (interquartile range) are Z. *54 patients were analyzed; †57 patients were analyzed; ‡54 patients were analyzed; §57 patients were analyzed. AE: Adverse event; ANGONG TRIAL: Safety and efficacy of Angong Niuhuang Pills in patients with moderate-to-severe acute ischemic stroke; ANP: Angong Niuhuang Pill; ECG: Electrocardiograph; SAE: Severe adverse event; –: Not applicable.
Discussion
The ANGONG TRIAL showed that ANP was safe in patients with acute moderate-to-severe ischemic stroke but was negative for primary and secondary outcomes. However, we found that ANP showed the potential to reduce infarct growth and improve the 90-day functional outcomes. Moreover, ANP significantly reduced infarct growth in ischemic stroke patients with LAA. This is a study to demonstrate the potential effect of ANP in reducing cerebral infarct volume in patients with acute moderate-to-severe ischemic stroke.
Previous studies reported that large infarct volume and infarct growth both predicted poor clinical outcomes.[7,30] Final infarct volume but not recanalization was independently associated with outcome.[30] Penumbra salvage volume also mediated the improved functional outcome after recanalization.[31] Infarct growth might be a potential therapeutic target in patients with AIS.[32] However, the infarct volume still increased from 14.8 mL at baseline to 37.5 mL at 24 h after EVT.[15] A randomized study assessed the efficacy of intravenous glyburide on large hemispheric infarct, but non-significantly increased proportion of the good outcome (mRS 0–4, 17% vs. 14%).[22] In our study, the baseline DWI infarct volume was >25 mL, similar to the lesion volume in patients receiving EVT.[15] A larger baseline infarct volume was assumed to indicate higher infarct growth.[33] We found ANP to be a potential candidate for reducing the infarct volume in patients with moderate-to-severe ischemic stroke, especially in those with LAA.
Cerebral edema often occurs as a complication of large infarcts and results in neurological deterioration. Swelling is a strong predictor of poor outcomes in non-lacunar stroke.[18] Reperfusion therapy, osmotic drugs, and intravenous glyburide have been used to reduce edema volume,[17,22,34] but their efficacy was poor. For patients with very large core volumes (>130 mL), EVT might increase the edema volume.[35] Reducing the volume of cerebral edema is another challenge for neurologists, as no effective therapy is recommended by current guidelines.[34] Our study did not confirm the conjecture of previous studies that ANP may reduce cerebral edema.[26] Further large-sample studies are needed to determine whether ANP reduces cerebral edema.
The ANGONG TRIAL included patients with baseline NIHSS scores between 10 and 20, indicating a higher risk of disability and mortality.[36,37] The rates of excellent outcome (mRS 0–1) were inversely associated with NIHSS score in those with or without alteplase.[10] Although EVT was beneficial to patients with LAA,[4] the real-world utilization rates of alteplase and EVT were only 10.2% and 4.9%, respectively.[38] The majority of patients with acute moderate-to-severe stroke did not receive reperfusion therapy and were at high risk of disability. More than half of the patients with NIHSS scores >10 were probably functionally dependent. In addition to alteplase and EVT, our study found that ANP could non-significantly improve the clinical outcome. In patients with LAA, ANP increased the rate of mRS 0–2 at 90 days from 24% to 39%, possibly because of the reduced volume of cerebral infarct.[39] Because of the correlation between infarct volume and functional prognosis, we speculated that ANP might improve the outcome of acute moderate-to-severe stroke, but further randomized controlled trials are warranted to confirm this result.
Exposure to ANP often raises safety concerns regarding heavy metals and their nephrotoxic damage, as realgar and cinnabar in ANP, contain 90% As2S3 and 96% HgS, respectively.[26,40] Previous studies reported low rates of AEs; however, the quality of these studies was poor.[26,41] In our study, the concentration of mercury and arsenic at day 7 did not increase in the ANP group. The frequency of SAE was lower in the ANP group than that in the placebo group. In addition, a systematic review reported that 85.2% of AEs were associated with original diseases, and two SAEs (2/49, 4.1%) occurred in children with an overdose of ANP.[27] Another predisposing factor for SAE was the use of ANP against the indications of traditional Chinese medicine (TCM).[27] We speculated that the exclusion of cases with contraindications to TCM may be one of the reasons for the low rate of SAE in our study.
Our study had several strengths. This randomized double-blind placebo-controlled study included patients with acute moderate-to-severe ischemic stroke. ANP was prescribed within 36 h of onset. The primary endpoints–cerebral infarct and edema volumes–were measured by an independent group of radiologists blinded to the treatment. Efficacy analysis was performed for both the FAS and PPS. To verify the safety issues of heavy metals, the concentrations of mercury and arsenic were tested centrally at baseline and day 7, though the safety of ANP has been reported in an animal study.[39] Before the trial initiation, we noticed that the main cause of ischemic stroke in patients with a NIHSS score of 10 or more was likely to be LAA,[42] and efficacy and safety analyses were also scheduled in patients with LAA. In addition, as a TCM, ANP has been widely used in clinical practice for more than one century in patients with unconsciousness due to heat blockage, based on the theory of TCM.[25,26,40] We, on the other hand, addressed the safety issue of ANP by the methodology of a randomized controlled trial,[26,41] and thus provided safety data for the following large-sample clinical study of ANP.
Our study had several limitations. Considering the exploratory nature of this pilot study, the sample size was set at 120. Power and type I errors were not calculated before trial initiation, and the rate of dropout was relatively high. In addition, we would like to remind investigators to focus on patient adherence when designing studies for severe ischemic stroke. The results of our study should be interpreted with caution. For the neurologists, radiological endpoints other than clinical outcomes were chosen as the primary outcomes, limiting the clinical significance of this study. However, cerebral infarct growth was also an acceptable intermediate indicator associated with a 90-day mRS score.[7,32] ANP also showed the potential to improve neurological function. Further studies are needed to confirm the efficacy of ANP in improving clinical outcomes. Collateral status and recanalization status were important factors influencing cerebral infarction and edema volume but were not included in our analysis.[17,33] Finally, we noticed that ANP might inhibit infarct growth 24 h after onset, but its effect on infarct volume increase within 24 h after onset was unclear.
In conclusion, treatment with ANP was safe but failed to significantly reduce cerebral infarct and edema volume. However, in patients with LAA, ANP marginally reduced the infarct volume. ANP also showed a tendency to reduce the volume of cerebral infarcts and increase the rate of functional independence, providing foundation for the following researches. These findings suggested the potential of ANP to reduce cerebral infarct volume improve clinical prognosis, and provided a scientific basis for further trials in patients with acute moderate-to-severe stroke.
Funding
This study was supported by the Projected Subjects of National Health Commission Stroke Prevention Project Expert Committee (No. GN-2020B0001) and Tongrentang Pharmaceutical Factory, Beijing Tongrentang Co.
Conflicts of interest
Lin Shi is the director of BrainNow Medical Technology Limited. Yishan Luo is now employed by BrainNow Medical Technology Limited. Other authors declare no competing interests.
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